exam questions and answers Flashcards
define denaturation
major change in a protein by application of external stress or compound
describe the bonds involved in the four levels of protein structure
primary - peptide bonds
secondary - hydrogen bonds
tertiary - hydrogen bonding, disulphide bridges, ionic interactions, hydrophobic interactions, van der waals
quaternary - all previous bonds
during denaturation, which level of structure will be disrupted and in what way?
quaternary - polypeptide chains separate
tertiary - all bonds break except disulphide bridges
secondary - hydrogen bonds break
primary - structure remains intact
describe the effect of temperature on proteins
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
how does pH bring about denaturation?
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
what is the function of a protease?
an enzyme that breaks down proteins into peptides.
discuss how trypsin can act as a chemical denaturant
it cleaves the proteins on the carboxyl side of lysine and arginine except when followed by proline
define proteolysis
directed degradation or digestion of proteins by proteases or intermolecular digestion
what is the role of a protease inhibitor cocktail?
designed to inhibit all protease enzymes and therefore limit degradation
which two chemicals can preserve cysteine residues, in what way do they do this?
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.
what are the conditions of storage used to maintain a protein in its native state?
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
define the term ‘binding site’
a region on a protein to which specific ligands form chemical bonds
describe chemical specificity
a measure of the types of ligands that will bind
describe affinity
measure of the strength of the chemical bond
there are 2 general characteristics that apply to all binding sites - what are they?
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
the binding of a ligand to its protein occurs through non-covalent interactions of which there are 4 main types - what are they?
hydrogen bonds
ionic bonds
hydrophobic interactions
van der waals forces
if molecules are found to have the same evolutionary origin, what does this tell us about their amino acid sequence?
they will have significant sequence similarity in their conserved regions
explain the principles of X-ray crystallography
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
knowledge of what level or protein structure is required for X-ray crystallography?
primary structure
describe why methotrexate is useful as a cancer drug
it inhibits the synthesis of purines and pyrimidines which are needed in rapidly dividing cells for DNA replication
discuss the steps involved in chemical modification of binding sites using the example of chymotrypsin
DIFP phosphorylates serine 195, this is an irreversible modification as it is stable to hydrolysis this inactivates the enzyme
discuss how pH dependence of activity is used to locate amino acid residues at binding sites
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.
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.
the binding of regulatory molecules to the allosteric site induces a conformational change which then affects the binding at the active site
allosteric proteins show ‘co-operativity’, what does this mean?
activity at one functional site affects the activity at others
modulator molecules can regulate protein activity through binding to the allosteric site. describe the difference between a positive modulator and a negative modulator.
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
explain the term cooperativity in relation to oxygen binding to haemoglobin
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.
what is phosphorylation?
the addition of a phosphate group to a Serine, Threonine, or Tyrosine residue on protein molecules through a condensation reaction
name the group of enzymes used in phosphorylation
kinases
what is the residue most commonly phosphorylated in proteins?
serine
phosphorylation results in the addition of two negative charges to the modified protein. what is the consequence of this?
new electrostatic interactions can be formed resulting in altered substrate binding and catalytic activity.
what is the primary determinant of specificity of multifunctional kinases?
the amino acid sequence surrounding the serine or threonine phosphorylation site
how to protein phosphatases reverse the effects of kinases?
catalyses the hydrolytic removal of phosphate groups from the proteins
describe the action of G-protein linked receptors using the example of the adenylyl cyclase system
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
which second messenger is used in the adenyly cyclase system?
cAMP
describe the function of protein kinases A
to phosphorylate target proteins making them active
describe in detail how protein kinase A can be activated
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
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?
cyclins
what is the function of CDK’s within the cell cycle?
to phosphorylate target proteins to orchestrate coordinated entry into the next phase of the cell cycle.
what is a zymogen?
the inactive pre-cursor of a protein
chymotrypsin is a digestive enzyme that hydrolyses protein in the small intestine. describe how it can be converted into its active form.
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)
what does SDS-PAGE stand for?
sodium dodecyl sulphate poly acrylamide gel electrophoresis
explain how you would determine if a protein was under control by a phosphorylation cascade?
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
define proteomics?
study of an organisms complete complement of proteins
name the 5 stages used in determining amino acid sequence in a protein
- sample preparation
- separation
- ionisation
- mass spectrometry
- informatics
explain the process of 2d gel electrophoresis?
1st dimension - pH gradient applied, proteins move to their isoelectric point
2nd dimension - separation proteins according to size using SDS-PAGE
define the isoelectric point
the pH at which an amino acid is electrically neutral so it will not migrate in an electric field.
what name is given to the group of enzymes used to cleave protein molecules?
proteases
what does each peak in a mass spectrometry plot represent?
an amino acid
what does MALDI-TOF stand for?
matrix assisted laser desorption ionisation time of flight
what does antibody trapping involve?
protein to be analysed is purified which allows antibodies to be raised so now we have antibodies plus protein of interest.
what process is used to separate the antibody-protein complexes? how are the proteins then identified?
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.
FRET allows us to see if protein associate with one another through the use of fluorescence, explain how this technique works.
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.
name and describe a chemical method of locating amino acid residues in binding sites.
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.
name and describe a structural method of locating amino acid residues in binding sites.
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.
give a description of FRET
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.
