proteins

Question 1

basic structure of a polypeptide

Answer 1

N terminus, C terminus
peptide bonds
amino acid side chains which dictate function

Question 2

ionic / electrostatic interactions in proteins

Answer 2

strong but do not greatly stabilise structure

Question 3

define salt bridge in proteins

Answer 3

association of two ionic protein groups of opposite charge = ion pair = salt bridge

Question 4

H bonds in proteins typically form between

Answer 4

N-H and C=O groups

Question 5

hydrophobic forces in proteins

Answer 5

important in maintaining protein stability
folds such that hydrophobic core region contains non-polar side chains, polar chains on the outside

Question 6

what is required to form disulphide bonds?

Answer 6

an OXIDISING ENVIRONMENT
cannot happen inside a cell

forms between thiol containing side chains (cysteines)

Question 7

what factors may cause a protein to denature?

Answer 7

change in temp, pH
detergents / organic substances / chaotropic agents

Question 8

define chaotropic agents, give examples

Answer 8

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

Question 9

how does pH affect a protein?

Answer 9

amino acids are zwitterionic
different pH can cause different charges, which are often involved in active site of enzymes

Question 10

how to produce / purify a protein

Answer 10

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

Question 11

name three different types of column chromatography to isolate proteins

Answer 11

1. ion exchange chromatography
2. gel-filtration
3. affinity

Question 12

how does ion exchange chromatography work

Answer 12

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.

Question 13

how does gel filtration chromatography work

Answer 13

inert porous matrix - molecules small enough to penetrate pass slower, larger molecules pass quicker

Question 14

how does affinity chromatography work

Answer 14

insoluble matrix linked to specific ligand which binds a specific protein, others wash through. can be eluted after to release protein.

Question 15

what is the isoelectric point?

Answer 15

the pH at which a protein has no net charge (usually have net charges due to amino acids)

Question 16

how is electrophoresis used to separate proteins?

Answer 16

electric field applied to sun of protein molecule, causing it to migrate cathode–>anode at a rate that depends on its SIZE and CHARGE.

Question 17

what is proteomics?

Answer 17

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

Question 18

how is peptide mass fingerprinting done?

Answer 18

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

Question 19

method to determine protein concentration

Answer 19

optical methods - certain amino acids absorb at different wavelengths, used to determine protein concentration

Question 20

Beer-lambert law

Answer 20

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

Question 21

method to determine 3D structure of proteins

Answer 21

X-ray crystallography
crystallising proteins is often challenging