proteins Flashcards
basic structure of a polypeptide
N terminus, C terminus
peptide bonds
amino acid side chains which dictate function
ionic / electrostatic interactions in proteins
strong but do not greatly stabilise structure
define salt bridge in proteins
association of two ionic protein groups of opposite charge = ion pair = salt bridge
H bonds in proteins typically form between
N-H and C=O groups
hydrophobic forces in proteins
important in maintaining protein stability
folds such that hydrophobic core region contains non-polar side chains, polar chains on the outside
what is required to form disulphide bonds?
an OXIDISING ENVIRONMENT
cannot happen inside a cell
forms between thiol containing side chains (cysteines)
what factors may cause a protein to denature?
change in temp, pH
detergents / organic substances / chaotropic agents
define chaotropic agents, give examples
ions that tend to denature proteins by increasing the solubility of non polar substances in water (disrupt hydrophobic interactions)
eg. guanidinium, urea
often used to REVERSIBLY denature proteins
how does pH affect a protein?
amino acids are zwitterionic
different pH can cause different charges, which are often involved in active site of enzymes
how to produce / purify a protein
recombinant plasmid DNA introduced to bacterial cell, which produces proteins of interest
1. lysis - eg. by freeze/thaw cycles
2. centrifuge - to isolate a particular part of the cell
3. chromatography - isolate protein
name three different types of column chromatography to isolate proteins
- ion exchange chromatography
- gel-filtration
- affinity
how does ion exchange chromatography work
matrix of column carries ionic charges, slowing down movement of molecules of opposite charge. molecules of the same charge pass quicker and are collected first.
how does gel filtration chromatography work
inert porous matrix - molecules small enough to penetrate pass slower, larger molecules pass quicker
how does affinity chromatography work
insoluble matrix linked to specific ligand which binds a specific protein, others wash through. can be eluted after to release protein.
what is the isoelectric point?
the pH at which a protein has no net charge (usually have net charges due to amino acids)
how is electrophoresis used to separate proteins?
electric field applied to sun of protein molecule, causing it to migrate cathode–>anode at a rate that depends on its SIZE and CHARGE.
what is proteomics?
the large-scale study of proteins -
done by separating proteins in a pH gradient (by isoelectric point) and then by their size.
very high resolution technique
how is peptide mass fingerprinting done?
couples proteomics with mass spectrometry.
purified proteins subject to proteolytic digestion using an enzyme that cuts peptide bonds (eg. trypsin), producing garments that are loaded into a mass spectrometer
used to construct a partial amino acid sequence
method to determine protein concentration
optical methods - certain amino acids absorb at different wavelengths, used to determine protein concentration
Beer-lambert law
used to determine conc of species that absorbs light
A (absorbance) = εcl
ε: molar extinction coefficient = how strongly a substance absorbs light at a given wavelength, per molar conc
c = conc
l = path length
method to determine 3D structure of proteins
X-ray crystallography
crystallising proteins is often challenging
