exam questions and answers

Question 1

define denaturation

Answer 1

major change in a protein by application of external stress or compound

Question 2

describe the bonds involved in the four levels of protein structure

Answer 2

primary - peptide bonds
secondary - hydrogen bonds
tertiary - hydrogen bonding, disulphide bridges, ionic interactions, hydrophobic interactions, van der waals
quaternary - all previous bonds

Question 3

during denaturation, which level of structure will be disrupted and in what way?

Answer 3

quaternary - polypeptide chains separate
tertiary - all bonds break except disulphide bridges
secondary - hydrogen bonds break
primary - structure remains intact

Question 4

describe the effect of temperature on proteins

Answer 4

when temperature increases above the optimum (40ºC) there will be 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

Question 5

how does pH bring about denaturation?

Answer 5

changes in pH will affect acidic and basic groups, this affects the equilibrium point of ionisation, this renders the protein inactive but maintains its 3D arrangement

Question 6

what is the function of a protease?

Answer 6

an enzyme that breaks down proteins into peptides.

Question 7

discuss how trypsin can act as a chemical denaturant

Answer 7

it cleaves the proteins on the carboxyl side of lysine and arginine except when followed by proline

Question 8

define proteolysis

Answer 8

directed degradation or digestion of proteins by proteases or intermolecular digestion

Question 9

what is the role of a protease inhibitor cocktail?

Answer 9

designed to inhibit all protease enzymes and therefore limit degradation

Question 10

which two chemicals can preserve cysteine residues, in what way do they do this?

Answer 10

DTT or 2ME, both reduce the disulphide bonds within the proteins, breaking down the tertiary structure of the protein which helps to preserve protein in a reduced state for analysis.

Question 11

what are the conditions of storage used to maintain a protein in its native state?

Answer 11

low temperature - this will slow down the activity of any protease enzymes present which would slow the degradation of proteins.
use of 50% glycerol or DMSO - this acts as a cryoprotectant and limits the formation of ice crystals in the protein

Question 12

define the term ‘binding site’

Answer 12

a region on a protein to which specific ligands form chemical bonds

Question 13

describe chemical specificity

Answer 13

a measure of the types of ligands that will bind

Question 14

describe affinity

Answer 14

measure of the strength of the chemical bond

Question 15

there are 2 general characteristics that apply to all binding sites - what are they?

Answer 15

crevices in the 3D structure where a ligand can form temporary non-covalent chemical bonds with, as it has complementary shape
the site excludes water so is surrounded by non-polar amino acids

Question 16

the binding of a ligand to its protein occurs through non-covalent interactions of which there are 4 main types - what are they?

Answer 16

hydrogen bonds
ionic bonds
hydrophobic interactions
van der waals forces

Question 17

if molecules are found to have the same evolutionary origin, what does this tell us about their amino acid sequence?

Answer 17

they will have significant sequence similarity in their conserved regions

Question 18

explain the principles of X-ray crystallography

Answer 18

involves bombarding a crystal of the protein with X-rays and then analysing the resultant diffraction pattern caused by the atoms in the protein scattering the X-rays.
the diffraction pattern produces an electron density map which with knowledge of the proteins primary structure is used to determine the 3D structure

Question 19

knowledge of what level or protein structure is required for X-ray crystallography?

Answer 19

primary structure

Question 20

describe why methotrexate is useful as a cancer drug

Answer 20

it inhibits the synthesis of purines and pyrimidines which are needed in rapidly dividing cells for DNA replication

Question 21

discuss the steps involved in chemical modification of binding sites using the example of chymotrypsin

Answer 21

DIFP phosphorylates serine 195, this is an irreversible modification as it is stable to hydrolysis this inactivates the enzyme

Question 22

discuss how pH dependence of activity is used to locate amino acid residues at binding sites

Answer 22

changing pH affects acidic/basic side chains and can be used to investigate if these are involved in binding sites. comparison of activity before and after pH changes will show if they are involved.

Question 23

Allosteric proteins contain distinct regulatory sites and multiple function sites and can be controlled by small signal molecules. Describe in general terms how the binding of small molecules can regulate protein activity.

Answer 23

the binding of regulatory molecules to the allosteric site induces a conformational change which then affects the binding at the active site

Question 24

allosteric proteins show ‘co-operativity’, what does this mean?

Answer 24

activity at one functional site affects the activity at others

Question 25

modulator molecules can regulate protein activity through binding to the allosteric site. describe the difference between a positive modulator and a negative modulator.

Answer 25

a positive modulator can bind to the allosteric site and so increase the affinity of the enzyme for its substrate
a negative modulator can bind to a different allosteric site and so reduce the affinity of the enzyme for its substrate

Question 26

explain the term cooperativity in relation to oxygen binding to haemoglobin

Answer 26

binding of oxygen to one subunit induces a conformational change which increases the affinity of the next subunit of haemoglobin to oxygen. this process is repeated with the binding of oxygen becoming increasingly easier.

Question 27

what is phosphorylation?

Answer 27

the addition of a phosphate group to a Serine, Threonine, or Tyrosine residue on protein molecules through a condensation reaction

Question 28

name the group of enzymes used in phosphorylation

Answer 28

kinases

Question 29

what is the residue most commonly phosphorylated in proteins?

Answer 29

serine

Question 30

phosphorylation results in the addition of two negative charges to the modified protein. what is the consequence of this?

Answer 30

new electrostatic interactions can be formed resulting in altered substrate binding and catalytic activity.

Question 31

what is the primary determinant of specificity of multifunctional kinases?

Answer 31

the amino acid sequence surrounding the serine or threonine phosphorylation site

Question 32

how to protein phosphatases reverse the effects of kinases?

Answer 32

catalyses the hydrolytic removal of phosphate groups from the proteins

Question 33

describe the action of G-protein linked receptors using the example of the adenylyl cyclase system

Answer 33

ligand binds to the transmembrane GPCR
the GPCR undergoes a conformational change activating the G protein
GDP is replaced by GTP in the G-protein
G-protein activates the effector - adenylyl cyclase
effector produces the second messenger cAMP

Question 34

which second messenger is used in the adenyly cyclase system?

Answer 34

cAMP

Question 35

describe the function of protein kinases A

Answer 35

to phosphorylate target proteins making them active

Question 36

describe in detail how protein kinase A can be activated

Answer 36

PKA exists in the R2C2 form
4 molecules of cAMP bind to the R subunits
release of the catalytic subunits
enzyme is active and phosphorylates target proteins

Question 37

movement through the cell cycle from one stage to the next depends on CDK’s. which group of molecules activate the catalytic activity of CDK’s?

Answer 37

cyclins

Question 38

what is the function of CDK’s within the cell cycle?

Answer 38

to phosphorylate target proteins to orchestrate coordinated entry into the next phase of the cell cycle.

Question 39

what is a zymogen?

Answer 39

the inactive pre-cursor of a protein

Question 40

chymotrypsin is a digestive enzyme that hydrolyses protein in the small intestine. describe how it can be converted into its active form.

Answer 40

inactive chymotrypsin (245 amino acids) is converted to pi chymotrypsin by action of trypsin.
pi chymotrypsin (2 active peptides) then act on other pi chymotrypsin to form alpha chymotrypsin (3 single chains, A,B and C)

Question 41

what does SDS-PAGE stand for?

Answer 41

sodium dodecyl sulphate poly acrylamide gel electrophoresis

Question 42

explain how you would determine if a protein was under control by a phosphorylation cascade?

Answer 42

run a target protein on SDS gel along with the target protein which has phosphate added to it
if the original target protein was phosphorylated, then the heavier protein will travel less distance on the gel

Question 43

define proteomics?

Answer 43

study of an organisms complete complement of proteins

Question 44

name the 5 stages used in determining amino acid sequence in a protein

Answer 44

1. sample preparation
2. separation
3. ionisation
4. mass spectrometry
5. informatics

Question 45

explain the process of 2d gel electrophoresis?

Answer 45

1st dimension - pH gradient applied, proteins move to their isoelectric point
2nd dimension - separation proteins according to size using SDS-PAGE

Question 46

define the isoelectric point

Answer 46

the pH at which an amino acid is electrically neutral so it will not migrate in an electric field.

Question 47

what name is given to the group of enzymes used to cleave protein molecules?

Answer 47

proteases

Question 48

what does each peak in a mass spectrometry plot represent?

Answer 48

an amino acid

Question 49

what does MALDI-TOF stand for?

Answer 49

matrix assisted laser desorption ionisation time of flight

Question 50

what does antibody trapping involve?

Answer 50

protein to be analysed is purified which allows antibodies to be raised so now we have antibodies plus protein of interest.

Question 51

what process is used to separate the antibody-protein complexes? how are the proteins then identified?

Answer 51

affinity chromatography - any antibody protein complexes bind to the stationary phase of the column, the remaining components pass through. the desired complex is then eluted by addition of buffer. proteins identified with 2D gel electrophoresis and mass spectrometry.

Question 52

FRET allows us to see if protein associate with one another through the use of fluorescence, explain how this technique works.

Answer 52

dependent on one fluorescent molecule absorbing fluorescent energy from another.
two proteins of interest are tagged with complementary fluorescent molecules, if the tagged proteins interact then there will be a change in fluorescent wavelength.

Question 53

name and describe a chemical method of locating amino acid residues in binding sites.

Answer 53

chemical modification - amino acids that are suspected to be crucial to enzyme functioning are chemically modified. this chemical modification leads to an altered activity of the enzyme indicating modification has had an effect.
pH dependence of activity - changing the pH will affect acidic or basic side chains of amino acids, this can be used to investigate the roles of amino acid residues and their role in activity. comparing the activity or binding before and after changing the pH can show if the amino acid residue is critical to protein function.

Question 54

name and describe a structural method of locating amino acid residues in binding sites.

Answer 54

use of structural analogues - structural analogues are similair to the substrate that would normally bind but do not react with the protein in the usual way, they are non-hydrolysable. they are crucial for the role of developing understanding of interactions within binding sites.
X-ray crystallography - protein in a crystal form is bombarded with X-rays, the diffraction pattern produced by atoms scattering the X-rays can be analysed, knowledge of the primary structure is required to then determine the 3D structure.

Question 55

give a description of FRET

Answer 55

one fluorescent molecule will be able to absorb fluorescent energy emitted by another so proteins suspected of forming complexes are tagged with complementary fluorescent molecules, if protein interaction has occurred then there will be a change in fluorescent wavelength.